The common NOD2/CARD15 variant P268S in patients with non-infectious uveitis: a cohort study

Patients

Twenty-five unrelated Italian patients with an established diagnosis of ACU have been
included in the present study: the population was composed of 19 pediatric and 6 adults.
Patients were consecutively enrolled in 2012–2013 at paediatric hospital “Anna Meyer”
(Florence, Italy) and at Hospital “Santa Maria alle Scotte” (Siena, Italy). Inclusion
criteria were the presence of ACU and willingness to participate in the study. Written
consent was obtained by all adult patients or guardians. Data regarding age of onset,
type and localization of uveitis, associated diseases, and complications were collected
and recorded with a customized database. The analysis of the exon 4 of the NOD2/CARD15
gene was also performed on the DNA of 25 tuscanian controls.

Controls have been chosen between patients referred to our centers in the 2010–2014
period with an history of mechanical or functional joint pain and submitted to DNA
collection; these patients were age and sex-matched and were use as historical control
population. We selected patients and controls of tuscanian ancestry, according to
family history reported, in order to guarante a common genetic background.

DNA extraction and genotyping

Blood samples were collected in EDTA-containing tubes during routine venipuncture.
DNA was extracted using the High Pure PCR Template Preparation kit (Roche Applied
Science, France), according to manufacturer’s instructions. Genomic DNA was eluted
in sterile water, and stored at ?20 °C; the concentration of the eluted genomic DNA
was determined by spectrophotometry with the Nanodrop® ND-1000 (NanoDrop Technologies
INC, Wilmington, DE, USA). PCR reaction was conducted using primers designed with
the Primer3 software. Samples were amplified in a Eppendorf Mastercycler thermal cycler
and then the PCR products were purified using the WizardSVGel and PCR Clean-up System
(Promega). The purification was applied to both PCR product and extracts from agarose
gel. The sequencing reaction was performed with ABI 3730 DNA Analyzer (Applied Biosystems
Life Sciences Corporation, Carlsbad, CA, U.S.A.) and the electropherogram analysis
was performed through the software Sequencher v.4.2.2 (GeneCodes, USA).

Statistical analysis

Allelic and genotypic frequencies (CC, CT, TT) in cases and controls were calculated;
then differences between groups in terms of SNP variants frequencies were determined
with chi-square test and Fisher exact test with Monte Carlo simulations, when appropriate.
Since the number of subjects carrying a TT genotype was very low, the analysis was
performed also comparing frequency data in two groups: those carrying at least one
CARD 15 *T allele and those carrying the CC genotype. The Spearman rank correlation
test was used to determine correlation coefficients for different variables [gender,
age at diagnosis, uveitis duration, types of uveitis (granulomatous, and not-granulomatous,
anterior, posterior, pars-planitis, mono/bilateral), ANA positivity, HLA-B27 presence,
auto-immune associated diseases, eye complications]. The odds ratio (OR) and 95 %
confidence interval (95 % CI) were calculated. Non-parametric tests were used, where
necessary, due to the small size of our groups and to the skewness of our data. Levels
of p??0.05 were considered statistically significant. All analyses were performed on
SPSS package for Windows, version 13.0 (SPSS, Inc., Chicago, IL, USA).

Prevalence of NOD2/CARD15 variants

The P268S/SNP5 (SNP rs2066842) involves a C to T change resulting in a P to S amino
acid change; these common variation of the NOD2/CARD15 gene have been identified in
17 of our cases: 15 patients showed the polymorphism P268S/SNP5 (SNP rs2066842) as
heterozygous carriers while 2 patients were homozygous for the same polymorphism.
The polymorphism distribution in AUC resulted as follows: 6/25 CC (24 %), 17/25 CT
(68 %), 2/25 TT (8 %) [Table 1]. One patient carried two different mutations: the polymorphism P268S/SNP5 in heterozygous
and a variant on intron 3 (c647 18–16 TCT). We regarded the variation c647 as a non-functional
one and so we include this patient in the same group as the heterozygous carriers
of the P268S/SNP5 polymorphism. The incidence of P268S/SNP5 in our population was
compared with the incidence reported for 25 healthy historical controls, age and sex-matched.
According to genotype analysis, 14/25 controls (56 %) did not present this polymorphism,
10/25 (40 %) were heterozygous carrier and 1/25 (4 %) were homozygous. No evidence
of departure from Hardy-Weinberg equilibrium in controls was seen (p?=?0.65).

Table 1. Comparison of CARD 15* genotype frequencies (%) in Autoimmune Chronic Uveitis versus
controls

Statistical analysis for CARD 15 genotyping showed significant differences between
patients and controls for allelic frequencies but not for genotypic frequencies. T
allele was present in 19/25 (76 %) AUC subjects compared to 11/25 (44 %) controls,
and C allele in 23/25 (33.3 %) (p?=?0.04, OR: 4.03, 95 % CI?=?1.2–13.5) [Table 2].

Table 2. A Comparison of CARD 15* C/T allele frequencies (%) in Autoimmune Chronic Uveitis
versus controls

The functional role of the P268S/SNP5 (SNP rs2066842) variant was predicted using
SIFT (version 5.0.2) and PolyPhen (PolyPhen-2 version 2.2.2, release 405).

Genotype-phenotype correlation

Table 3 show the main characteristics of our cohort of enrolled patients. We also tried to
assess if the T allele presence could impact the clinical course of the disease in
patients. We failed to show any association between one genotype and the considered
clinical variables. In particular, outcome of ACU was not modified by NOD2/CARD15
polymorphisms, since the incidence of macular edema, synechiae, glaucoma, visual loss
and cataract was not increased in the group of patients heterozygous or homozygous
for P268S/SNP5 [Table 4].

Table 3. Demographical and medical characteristics of the studied population

Table 4. Phenotype-genotype correlation